298 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
ion to the essential N 2 , N 1 , or O 6 positions of the G 5.0 guanosine. Metal ion
interactions are almost never seen at the essential positions at the cleavage
site — that is, the pro - R p - oxygen of A 1.1 or the ribose O2 ′ or O 2 positions of
C17.0. One interaction in the PDB: 301D structure places a Mg 2+ within 2.4 Å
of the pro - S p - oxygen of A 1.1 , an atom not found to be essential in biochemical
or spectroscopic experiments.
Blount and Uhlenbeck^61 state that the global hammerhead fold in the
crystal structures agrees with studies carried out in solution including fl uores-
cence resonance energy transfer (FRET), native gel electrophoresis, transient
electric birefringence, and nuclear magnetic resonance (NMR) experiments.
Some of these have been discussed previously in this section. However, atomic -
level detail of a hammerhead solution structure, especially in the core region,
has not been possible. Probably this is due to the highly dynamic nature of the
core structure in solution that likely exists as a family of rapidly interconvert-
ing different conformations. In fact, a^1 H - and^31 P - NMR study showed that the
core region near the essential pro - R p - oxygen of A 9 was inherently fl exible and
that this fl exibility could be important for hammerhead cleavage.^62
In the reference 61 review, the authors establish criteria for evaluation of
the many experiments that attempt to defi ne the essential functional groups
for hammerhead catalysis. These are as follows: (1) The measured cleavage
rate must refl ect the chemical step of the reaction, k 2 , and not a conformational
change, formation of the substrate – ribozyme complex, or release of the ribo-
zyme – product complex; (2) the hammerhead construct used must have accept-
able kinetic properties; and (3) the chemical nature of the functional group
modifi cation should not, by itself, cause conformational or mechanistic
changes — that is, the p Ka for the reactions should be the same before and after
the modifi cation. To measure k 2 successfully, one must measure the cleavage
rate under single - turnover conditions. Kinetically well - behaved hammerheads
are hammerheads 6, 8, and 16 (see Figures 6.10 , 6.11 , and 6.12 ). In most cases,
only one modifi cation to a nucleotide should be undertaken at a time; other-
wise, it is diffi cult to pinpoint the exact nucleotide position causing observed
changes. By evaluating available biochemical data according to these criteria,
the reference 61 authors established a consensus set of functional groups
unambiguously important for hammerhead catalysis. These are collected in
Figure 4 and Table 2 of reference 61. If one maps the collected important
functional groups — essentially from those residues defi ned as belonging to
domain I and domain II — one fi nds a set of atoms lining the catalytic pocket
starting at G 10.1 in domain II (relatively far from, or distal to, the catalytic site)
and ending at the cleavage site, C 17.0 , in domain I. The mapping exercise helps
in visualizing why nearby essential functional groups could have important
catalytic effects but raises questions as to why far - away functional groups (up
to 20 Å away) in domain II should have > 100 - fold effects on catalytic cleavage.
The review authors fi nd 21 essential functional groups in domain II and 13 in
domain I. Only four 2 ′ - hydroxyls are essential — those at G 5 , G 8 , C 16.1 , and C 17.
Only four phosphate oxygens appear to be essential — those 5 ′ to X 1.1 , A 9 , A 13 ,